From conformation to chemistry

Drug design has classically been represented as creating a key, a drug, that fits perfectly into a lock, the targeted protein. However, as our understanding of proteins’ dynamic nature has grown, scientists have gained insight into options for targeting them — new ways to try opening the lock. 

At St. Jude, researchers explore in detail how small molecules, like natural ligands and drugs, bind their targets. However, these interactions do not happen in a vacuum. Drug–protein binding creates a chain reaction, starting at the first amino acid contact and carrying through an entire cellular pathway. Understanding the nuanced conformational changes that occur within drug targets is vital to the design of next-generation therapeutics.

M. Madan Babu, PhD, FRS, Center of Excellence for Data-Driven Discovery director, and Department of Structural Biology member, leveraged data science, pharmacology, and structural information to study how each amino acid in the receptor that binds adrenaline contributes to receptor activity. Published in Science, the study revealed which amino acids control key pharmacological properties of the ligand. 

The adrenaline receptor studied here is a member of the G-protein–coupled receptor (GPCR) family. One-third of all U.S. Food and Drug Administration (FDA)–approved drugs target receptors in this family. Thus, understanding how GPCRs respond to natural or therapeutic ligands is critical for developing new therapies with precise effects on receptor activity. 

“Through evolution, every amino acid in the receptor has been sculpted in some way or another to ensure that it binds the natural ligand and elicits the appropriate physiological response,” said Babu. “It was exciting to discover the allosteric network governing GPCR function and to reveal that some amino acids control efficacy, some control potency, and others affect both.

The researchers developed a data science framework to integrate pharmacological and structural data systematically and revealed the first comprehensive picture of GPCR signaling. “When we mapped the pharmacological data onto the structure, they formed a beautiful network,” said Babu. 

“It provided new insights into the allosteric network linking the ligand binding pocket to the G protein binding site that governs efficacy and potency,” added Brian Kobilka, MD, co-corresponding author and the 2012 Nobel Prize winner in Chemistry from Stanford University School of Medicine.  

By understanding GPCR signaling at the atomic level, the researchers are optimistic that they can more closely observe the transient sub-states between the active and inactive conformations and thoroughly explore the proteins’ greater conformational landscape. 

While GPCRs have long been a focus of drug discovery due to their well-defined binding sites and broad function, nuclear receptors have recently gained attention as potential therapeutic intervention routes. 

Nuclear receptors are a family of 48 human proteins that regulate gene activation in response to signaling molecules. Their myriad versions (isoforms) comprise two distinct binding sites: a ligand-binding domain and a DNA-binding domain. Ligand binding acts as a signal for the receptor to move inside the cell’s nucleus. The DNA-binding site is naturally designed to then bind and activate a particular set of genes.

Although some noticeable trends exist, the range of genomic sites where nuclear receptors bind is confounding. Despite containing the same genome, a given nuclear receptor can bind at different genomic locations in different cells. 

“Same genome, same binding sites. So, wlohy are these receptors binding in different places?” questioned Aseem Ansari, Department of Chemical Biology & Therapeutics chair.

In a paper published in Nature Communications, Ansari and his team explored the underlying DNA-sequence patterns that enable nuclear receptor binding. To do so, they collaborated with Parameswaran Ramanathan, PhD, an electrical and computer engineer at the University of Wisconsin-Madison who uses a pattern-finding approach to design electrical circuits. 

This process involves finding the shortest route with the minimum input necessary to perform a function. This collaboration resulted in the development of MinSeq Find, a search algorithm designed to solve, at the single-nucleotide level, the masked DNA-sequence features necessary for nuclear receptors to bind DNA. 

They found a finely tuned process driven by not only the DNA sequence but also the initial ligand-binding restrictions. Such restrictions included whether one or two receptors are needed, the importance of communication between receptor-binding domains, and the structure of the DNA itself. These factors contributed to defining the different binding patterns observed across different cells.

The ligand-binding domains of nuclear receptors have unique therapeutic potential and pitfalls. Pregnane X receptor (PXR) functions in detoxification by activating genes that encode drug-metabolizing enzymes, utilizing its remarkably malleable ligand-binding site to detect a diverse panel of foreign chemicals, including chemotherapeutics. In a report published in Proceedings of the National Academy of Sciences (USA), Taosheng Chen, PhD, Department of Chemical Biology & Therapeutics, explored the rules governing the promiscuous nature of PXR and how to break those rules.

The researchers changed a drug that normally binds well to PXR into one that stretches the protein’s binding region, making binding energetically unfavorable. The modified drug lowered the levels of PXR-induced enzymes — indicating this approach could be used in drug development to evade the detoxification network. The potential implications of this research are vast; many drugs interact with PXR, and over half of all FDA-approved drugs in the U.S. are metabolized by PXR-induced enzymes.

In addition to identifying routes to reduce a drug’s potential to activate PXR, Chen is exploring targeting PXR directly for degradation. The degradation pathway involves proteins called E3 ubiquitin ligases; these enzymes label proteins for removal. A “hijacked” E3 can be used to degrade PXR. 

“One of our hypotheses is that maybe PXR is degraded by a certain E3 and that certain E3 might be downregulated in specific tissues like the liver, where PXR is highly expressed,” Chen explained. “There is potential to modulate selective PXR–E3 interactions to temporarily remove PXR from the equation.”

An example of such a PXR–E3 pair was published in Acta Pharmaceutica Sinica B, where researchers identified the F-box-only protein 44 assigned for PXR. The ability to target pathways such as this one would provide another avenue to block PXR-mediated drug metabolism.

The insight gained from a full conformational interrogation of drug targets has yet to be fully realized. Researchers no longer view these proteins as locks for which the correct key must be designed. Instead, St. Jude researchers continue to show that drug targets are complex networks for which our complete understanding is the key to unlocking their full therapeutic potential.